Isolation of neurones and stem cells for nerve fibres from a sample

ABSTRACT

The invention relates to the use of an L1 promoter-regulated protein, and L1-associated molecule, of L1, or of recognition molecules directed towards the above-mentioned structures, for the isolation of neurons and stem cells for nervous fibers, especially neural and/or neuronal stem cells, from a sample; the invention also relates to a method of isolating neurons and stem cells for nervous fibers using the above-mentioned recognition molecules. The fields of application of this invention are medicine and the pharmaceutical industry.

The invention relates to the use of an L1 promoter-regulated protein, anL1-associated molecule, of L1, or of recognition molecules directedtowards the above-mentioned structures, for the isolation of neurons andstem cells for nervous fibers, especially neural and/or neuronal stemcells, from a sample; the invention also relates to a method ofisolating neurons and stem cells for nervous fibers using theabove-mentioned recognition molecules. The fields of application of thisinvention are medicine and the pharmaceutical industry.

The central nervous system (CNS) is constituted of various types ofcells, with neurons, macroglia cells, such as astrocytes andoligodendrocytes, and microglia cells forming the most importantclasses. Neurons are important in the electrochemical transmission ofinformation in the nervous system, while microglia cells representhighly important components of the cellular immune response within theCNS, and macroglia cells mainly assume supporting functions (Jacobson,Developmental Neurobiology, New York: Plenum Press, 1991). Macrogliacells form a framework in the CNS, providing neurons with metabolites.More recent investigations have demonstrated that a variety ofadditional molecular interactions between macroglia cells and neuronstake place, which interactions enable CNS development and are alsoimportant for the function and plasticity thereof (Kettenmann & Ransom,Neuroglia, Oxford: Oxford University Press, 1995).

Most conveniently, such interactions can be investigated on culturedcells under well-defined conditions. One important precondition for suchinvestigations is to separate the neurons from other cells, so as toallow identification and testing of the direct effect of, in particular,glial, growth and differentiation factors on the neurons. To date, it ismerely possible to purify retinal gangliocytes from rats, using aso-called immunopanning procedure (Barres et al., 1998). This procedureutilizes the RGC-specific expression of the Thy-1 cell adhesion protein.Other neurons, particularly CNS neurons, cannot be isolated with thedesired purity and yield as yet.

The invention was therefore based on the object of isolating oraccumulating neurons and differentiated stem cells, in particularneuronal and/or neural stem cells, from a sample so as to make themuseful for various applications, for example.

The invention solves this technical problem by using an L1promoter-regulated protein, an L1-associated molecule, an L1 celladhesion protein, a recognition molecule directed towards a proteinregulated by an L1 promoter, towards an L1-associated molecule and/ortowards an L1 cell adhesion protein for the isolation of neurons anddifferentiated stem cells, particularly neural and/or neuronal stemcells, from a sample.

Accordingly, the teaching of the invention is based on the surprisingfinding that neurons and/or differentiated, particularly neural and/orneuronal stem cells can be selected or isolated specifically via the L1cell adhesion protein, via proteins regulated by the L1 promoter, andvia an L1-associated molecule, or via recognition molecules directedtowards the above-mentioned structures. In particular, L1 and theabove-mentioned L1 equivalents are expressed in the CNS, specifically byneurons. Surprisingly, it has also been possible to demonstrate that notonly neurons from the CNS, e.g. from the postnatal CNS, but also fromstem cells, especially differentiated stem cells, preferably neuronaldifferentiated adult or embryonal stem cells, can be isolated via L1 andL1 equivalents. In particular, this is advantageous in those cases wheree.g. embryonal stem cells undergo neural differentiation by addition ofsuitable factors. In addition to neurons, the resulting cell populationinvariably includes glia cells, and in some cases even other, non-neuraltypes of cells. Such a mixed population is unsuitable particularly intransplantation therapy because non-neuronal, i.e., dividing cellsinvolve the risk of developing into tumors after transplantation. Withthe aid of the teaching according to the invention it is possible forthe first time to obtain a purely neuronal cell population from stemcells, particularly for transplantation purposes, e.g. inneurodegenerative diseases. Furthermore, the neurons thus obtained orisolated can be used to investigate the conditions of neuronal growthand differentiation.

A sample in the meaning of the invention denotes any biological itemcollected by sampling, or a partial quantity or small amount thereof,the nature of which can be investigated by chemical, biological,clinical means or the like, particularly for the purpose of obtainingneurons or differentiated stem cells from such a sample; for example,the sample can be an accumulation of stem cells or a piece of a brain.More specifically, collecting or obtaining the sample is effected insuch a way that the collected partial quantity corresponds to an averageof the overall amount. The features determined by investigation or fromthe sample can be used—e.g. prior to isolating neurons or collectingstem cells—to assess the amount comprised by the sample, therebyallowing conclusions as to the overall amount. For investigation, thesamples can be pretreated by mixing, dividing, separating,pre-fractionating, reducing in size, adding enzymes or markers, or byother means. Various ways of pretreating the samples are known to thoseskilled in the art. Obviously, it may also be envisaged to collect thesample such that it does not correspond to an average of the overallamount. In particular, samples are biological materials comprisingstructures suitable for transmitting or storing information on abiological basis. For example, these include the CNS or parts thereof,the spinal cord, neocortex, striatum, etc. Furthermore, fluids, cellaccumulations or tissues comprising rod, cone and olfactory cells may beconcerned, as well as structures comprising amacrinal cells, horizontalcells, bipolar cells, Golgi cells, Purkinje cells, motoneurons, hair,pyramidal and/or basket cells. Accordingly, the sample may comprisenumerous other materials such as blood, lymph, urine, cerebrospinalfluid, liquids from bioreactors, lipid mixtures, bone, cartilage, andmany others.

Neurons in the meaning of the invention are all nervous cells which,being highly differentiated cells, are capable of receiving, processingand transmitting nervous stimuli. For example, these cells occur in theCNS, in ganglions and in sensory organs and may represent a partialquantity of the sample. Neurons or nervous cells in the meaning of theinvention consist of a cell body (=Corpus neuroni) and a nucleus whichmay be surrounded by a cytoplasm. In the meaning of the invention thecells themselves may be referred to as perikaryon. In the meaning of theinvention the nervous cell or neuron also has e.g. neurofibrils, Nisslsubstance and appendices, as well as one or more dendrites.

For example, the nervous cells can be nervous cells from animals orhumans. It is possible, for instance, to isolate adendritic nervouscells or apolar nervous cells which have no appendices, or bipolarnervous cells, as sensitive or sensory nervous cells having one polardendrite and neurite each, such as occurring in the form of granularcells of the retina, or multipolar nervous cells having severaldendrites and a long or short neurite, as motoric cells of the voluntaryand autonomic systems. Furthermore, it is possible to isolatepolyneuritic nervous cells having a plurality of neurites, e.g. as Cajalcells of the cerebral cortex or pseudo-unipolar nervous cells such asoccurring in sensitive ganglions of the head and spinal cord, orunipolar nervous cells wherein two appendices originally present areapparently fused into one, such as occurring in rod and cone olfactorycells, provided these cells can be subsumed under the term neurons inthe meaning of the invention. Of course, neuro-secretory nervous cells,as obtained e.g. from the hypothalamus, or neuromelanocytes may also beunderstood as neurons in the meaning of the invention. Depending ontheir location in the nervous system, the neurons can also be isolatedas preganglionic or post-ganglionic neurons and as interneurons. Othercells defined as nervous cells or neurons according to the inventionare, inter alia, amacrinal cells, horizontal cells, bipolar cells, Golgicells, Purkinje cells, motoneurons, hair, pyramidal and/or basket cells.

Amacrinal cells in the meaning of the invention are multipolar nervouscells, especially in the inner granular layer of the retina of the eye,which cells have short appendices. Horizontal cells are cells which,with their cell bodies containing nuclei, represent interneurons of theoptic pathway situated in the inner granular layer of the retina.Horizontal cells may also include the Cajal cells which occasionallyoccur in the uppermost layer of the cerebral cortex and represent smallspindle-shaped nervous cells with long, horizontally orientedappendices. Bipolar cells or bipolar neurons are nervous cells havingtwo appendices extending separately therefrom, and these cells occur inthe ganglions of the inner ear and of the retina. Golgi cells in themeaning of the invention are large granular cells of the cerebellarcortex with short neurites or cells to be subsumed under the termCellulae axiramificatae. Purkinje cells in the meaning of the inventionare large, pear-shaped nervous cells in the Stratum gangliosum of thecerebellar cortex, each having two to three dendrites extendingvertically into the molecular layer and branching in a plane therein andneurites descending into the cerebellar medulla. The motoneuron isunderstood to be the last neuron in the efferent innervation of theskeletal muscles, consisting of a ganglion cell situated in the anteriorhorns of the spinal cord and of the neurite innervating the nervouscells. The motoneuron forms a motoric unit of the skeletal muscle andconsists of an alpha-motoneuron with all muscle fibers innervated bythis neuron, each muscle fiber having only one motoric end plate. Haircells are understood to be auditory cells of the Corti apparatus. Theyare secondary sensory cells between the supporting cells of the Cortiorgan of the inner ear, provided with auditory hairs. They terminatesynaptically at the dendrites of the bipolar ganglion cells of theGanglion spirale. Pyramidal cells in the meaning of the invention arepyramid-shaped multipolar nervous cells of the cerebral cortex, theapical dendrites thereof extending into the molecular layer, while theshorter ones branch horizontally from the basal corners, and theneurites thereof extending from the cell basis towards the medulla.Basket cells in the meaning of the invention are asteroid nervous cellsin the molecular layer of the little brain, the abundant branchesthereof, formed by their neurites, braiding as basket fibers around thePurkinje cells.

In the meaning of the invention, nervous cells are particularly thosecells occurring in the CNS, and they can be referred to as neurons.According to the invention, however, the occurrence of these neurons isnot restricted to the CNS. For example, it is also possible that e.g.basket cells are modified by suitable factors to form cells havingneuron properties, in particular.

In addition to neurons or nervous cells or neurocytes, the teaching ofthe invention can also be used to isolate or separate or accumulatedifferentiated stem cells, e.g. neural and/or neuronal stem cells, froma sample. Inter alia, neurons, oligodendrocytes and astrocytes may beformed from the adult neural stem cells, especially from the centralnervous system. In humans, for example, these cells are predominantlyfound in the ventricular and subventricular zone of the brain and in thesubgranular zone of the Gyros dentatos in the hippocampus. The embryonalstem cells, for example, derive from the early phase of development ofan embryo prior to nidation, namely, from the so-called inner cell mass,i.e., the blastocyst. These cells are pluripotent, that is, they arecapable of differentiating into any tissue of the human organism. Unlikezygotes, however, they are not capable of developing into completeembryos. Similarly, these stem cells are capable of differentiating intoneurons in the meaning of the invention, thereafter being neurons in themeaning of the invention. However, they can be understood as such evenprior to differentiation. To use the above-mentioned stem cells e.g. intherapy, it is important that the cell preparation has high purity. Withthe aid of the teaching of the invention it is possible for the firsttime to separate the developing neurons from the insulin-producing cellsor blood cells developing simultaneously from the embryonal stem cells.The neurons obtained in this way can be used in transplantations, e.g.in Parkinson and Alzheimer patients.

Also, neuronal stem cells in the meaning of the invention are all thosecells that are known as stem cells from the nervous system or can befound therein. In the stem cell formation zone approximately one out ofthree hundred cells is a stem cell. For example, a cell population with80% purity can be obtained from an adult brain using automatic flowcytometry to provide an almost completely pure population of neuronalstem cells with the aid of the teaching of the invention, provided thestem cells exhibit L1, L1-associated structures or fragments orequivalents of such a structure, e.g. L1 homologues such as neuroglian.

Furthermore, the stem cells obtained, especially neuronal stem cells,can be stimulated—by means of factors known to those skilled in theart—in a way so as to form astrocytes and neurons or nervous cells; inthis process the simultaneous occurrence of muscle cells or other cellsin the culture can never be excluded. The cells thus obtained can beseparated, likewise using the method according to the invention,because—in the course of the individual development of the cells—thosecells developing as neurons can be separated from those having e.g. gliacell properties or those exhibiting properties of astrocytes. Theneuronal stem cells can be isolated from most various regions of theadult CNS, e.g. from the ventricular walls or from the striatum.Furthermore, they can be obtained from the outer germinal zone of thelittle brain of newborn mice. Other sources from which neuronal stemcells can be isolated, or stem cells which allow differentiation intoneuronal stem cells, are well-known to those skilled in the art.

Accordingly, stem cells in the meaning of the invention are bothembryonal and adult stem cells which, in particular, are already neuralor differentiated in a neuronal fashion, or allow differentiation tothis effect. Surprisingly, not only neurons but also differentiatedembryonal and adult stem cells, especially neuronal-differentiated stemcells, can be isolated via L1 or L1 equivalents. L1 equivalents in themeaning of the invention are all those structures interacting withspecific recognition molecules in a way so as to allow isolation ofneurons and differentiated stem cells. On the one hand, cell adhesionproteins L1 are understood to be neuronal cell adhesion moleculespertaining to the Ig superfamily in vertebrates and invertebrates. Inaddition to L1, the neuronal cell adhesion molecules—as is well-known tothose skilled in the art—include axonin, neuroglian, N-CAM, fascilin,DM-GRASP BEN SC1, IRRI C-RST, the polio virus receptor and others, whichare closely related to L1 and therefore can be understood as L1equivalents in the meaning of the invention. Homologies between celladhesion molecules of invertebrates and vertebrates, particularly on thelevel of the secondary and tertiary structures, e.g. between neuroglianand L1 and also, between fascilin and N-CAM and between the IRRS C geneproduct and DM-GRASP, are well-known to those skilled in the art. Inaddition to these structural common features of neuronal cell adhesionmolecules, i.e., in addition to the L1 equivalents in the meaning of theinvention, these moleculee are also capable of significantly promotingneurite growth. L1 in the meaning of the invention is particularlyeffective by stimulating in particular the neurite growth on Schwanncells by homophilic binding mechanisms between Schwann cells andneurons. Lesions of peripheral nerves give rise to increased expressionof L1 especially in neurons and Schwan cells, and L1 may also beassociated with axonal regeneration. L1 equivalents in the meaning ofthe invention may also include all proteins regulated by an L1 promoter.All those proteins or lipid or carbohydrate structures associated withthe L1 protein in a way so as to allow isolation of the above-mentionedstem cells and neurons are L1-associated molecules in the meaning of theinvention. Those skilled in the art are familiar with the fact thatL1-associated molecules may also be sugar or lipid structures, forexample, which associate with, e.g. bind to, L1 or L1 equivalents in away so as to allow the use thereof in the isolation of neurons, neuraland/or neuronal stem cells from a sample. Those skilled in the art arefamiliar with the fact that in addition to L1 and L1 equivalents orL1-associated molecules, it is also possible to use molecules having aclose relationship to neuroglian or L1, provided they occur in neuronsor in the above-mentioned stem cells. For example, these include thevertebrate molecule contactin wherein the number of FN III domains ismerely reduced by one. The above-mentioned L1 equivalents are preservedover a long period of time during the evolution of the nervous system,and therefore, a variety of molecules, especially proteins, but alsolipids or carbohydrates associated therewith, are available to thoseskilled in the art, which can be used in the meaning of the teaching ofthe present patent application. Obviously, L1 may also be a component ofa fusion protein.

In the meaning of the invention, isolation means that the collectedneurons or nervous cells or neuronal and/or neural stem cells can beobtained with a purity of 40%, preferably 50%, more preferable 60%, andespecially preferably 70, 80, 90, 95, 97, 99 and more than 99.5%.Various methods of isolating cells and determining the degree of purityare well-known to those skilled in the art.

In the meaning of the invention it is of course also possible to userecognition structures directed towards L1, L1-associated molecules orproteins regulated by the L1 promoter, i.e., all L1 equivalents in abroader sense, to isolate neurons and/or the above-mentioned stem cells.For example, immobilization of the recognition molecules, e.g. anantibody against the cell adhesion protein L1, on a culture dish or in achromatography column filled e.g. with nylon wool, can be envisaged.Now, when contacting a cell suspension or a sample including the neuronsor the above-mentioned stem cells with said culture dish orchromatography column, the structures to be isolated can interact withthe recognition molecules such that all those cells lacking theabove-mentioned L1 equivalents can be separated by washing or othermethods known to those skilled in the art from the neurons or stem cellsto be isolated.

In a preferred embodiment of the invention the recognition molecule isan antibody, an aptamer, a lectin, an antisense construct, a selectivechelator, fragments and/or a combination thereof. Antibody denotes apolypeptide encoded substantially by an immunoglobulin gene or byimmunoglobulin genes or fragments thereof specifically binding andrecognizing an analyte, i.e., L1, L1-associated molecules, proteinsregulated by an L1 promoter, that is, L1 equivalents. Well-knownimmunoglobulin genes include the kappa-, lambda-, alpha-, gamma-,delta-, epsilon- and mu-genes for the constant region, as well as theinnumerable genes for the variable immunoglobulin region. For example,antibodies can be intact immunoglobulin or a number ofwell-characterized fragments produced by cleavage with variouspeptidases. Antibody also denotes modified antibodies (e.g. oligomers,reduced, oxidized and labelled antibodies). The term antibody as used inthe present specification also encompasses antibody fragments producedeither by modification of whole antibodies or by de novo synthesis usingrecombinant DNA techniques. The term antibody encompasses both intactmolecules and fragments thereof, such as Fab, F(ab′)2 and Fv, capable ofbinding the epitope determinant. In these fragments the auto-antibody'scapability of selectively binding its antigen or the L1 equivalent isretained in part, the fragments being defined as follows:

-   (1) Fab, the fragment which contains a monovalent antigen-binding    fragment of an antibody molecule, can be generated by cleavage of a    whole antibody using the enzyme papain, thereby obtaining an intact    light chain and part of a heavy chain;-   (2) the Fab′ fragment of an antibody molecule can be produced by    treatment of a whole antibody with pepsin and subsequent reduction,    thereby obtaining an intact light chain and part of a heavy chain;    two Fab′ fragments per antibody molecule are obtained;-   (3) F(ab′)₂, the fragment of the antibody, which can be obtained by    treatment of a whole antibody with the enzyme pepsin without    subsequent reduction; F(ab′)₂ is a dimer comprised of two Fab′    fragments held together by two disulfide bonds;    -   (4) Fv, defined as a fragment modified by genetic engineering,        which includes the variable region of the light chain and the        variable region of the heavy chain and is expressed in the form        of two chains; and    -   (5) single-chain antibody (ScA), defined as a molecule modified        by genetic engineering, which includes the variable region of        the light chain and the variable region of the heavy chain        linked by a suitable polypeptide linker to form a genetically        fused single-chain molecule.

Methods of producing these fragments are well-known in the art (cf. e.g.Harlow and Lane, Antibodies: A Laboratory Manual, (1988), Cold SpringHarbor Laboratory, New York).

The term epitope as used in the present invention represents any antigendeterminant on antigens, particularly those associated with L1,L1-associated molecules such as sugar residues or proteins regulated byan L1 promoter, to which the paratope of an antibody binds. Epitopedeterminants normally consist of chemically active surface groups ofmolecules such as amino acids or sugar side-chains and normally havespecific features of the three-dimensional structure, as well asspecific charge properties.

Those skilled in the art can easily produce monoclonal antibodies in themeaning of the invention, directed against the inventive proteins andfragments thereof or other biological structures. General methods ofproducing Monoclonal antibodies using hybridoma techniques arewell-known. Immortalized cell lines producing antibodies can begenerated using cell fusion as well as other methods such as directtransformation of B lymphocytes with oncogenic DNA or transfection withEpstein-Barr virus. See, for example, M. Schreier et al., “HybridomaTechniques” (1980); Hammerling et al., “Monoclonal Antibodies and T-cellHybridomas” (1981); Kennet et al., “Monoclonal Antibodies” (1980); seealso U.S. Pat. Nos. 4,341,761, 4,399,121, 4,427,783, 4,444,887,4,452,570, 4,466,917, 4,472,500, 4,491,632, and 4,493,890. Groups ofmonoclonal antibodies or fragments thereof directed against the proteinof interest can be screened with respect to various properties, namely,isotope, epitope, affinity, etc. In another embodiment, genes encodingmonoclonal antibodies of interest can be isolated from hybridomas usingPCR methods well-known in the art, cloned into suitable vectors, andexpressed. When using immunoaffinity methods, monoclonal antibodies aresuited to purify the individual cell comprising proteins, such as L1equivalents, which they are directed against. Regardless whethermonoclonal or polyclonal antibodies are concerned, the antibodiesaccording to the invention are useful in that they can be used asreagents in immunoassays such as RIA, ELISA and the like. Furthermore,they can be used in the isolation of the L1 equivalents or of domainsfrom cells or of other biological samples. For example, the antibodiescan be used to establish an assay based on a tissue culture to find,isolate or modify new L1 antigens or new compounds that modify theinteraction between L1 antigens and receptors and/or target sites.

Humanized or chimeric antibodies may include portions derived from twodifferent species (e.g. human constant region and mouse binding region).The portions derived from two different species can be chemically linkedusing conventional methods, or can be produced as a single fusionprotein using methods of genetic engineering. A DNA encoding theproteins of both portions of the chimeric antibody can be expressed as asingle fusion protein.

An antibody binds specifically to a protein, e.g. another biologicalstructure, or exhibits specific immunoreactivity in this way if theantibody assumes its function in a binding reaction in the presence of aheterogeneous population of proteins and other biological substances,which reaction allows a decision to be made whether the protein oranother biological structure is present and, in particular, whether thecells comprising the proteins can be isolated. Under the establishedconditions of an immunoassay, the antibodies mentioned preferably bindto a specific protein, whereas no significant binding to other proteinspresent in the sample takes place. Specific binding to a protein undersuch conditions requires an antibody which has been selected accordingto its specificity for a particular protein, such as L1. Variousvariants of immunoassays can be used to select antibodies havingspecific immunoreactivity with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies having specific immunoreactivity with a particular protein. Adescription of immunoassay variants and conditions which can be used todetermine a specific immunoreactivity can be found in Harlow and Lane,Antibodies; A Laboratory Manual (1988), Cold Spring Harbor Publications,New York.

Preferably, the antibody is a single-chain antibody, a multi-body, a Fabfragment, an MHC molecule, an MHC peptide, a fusion peptide, a mimicrypeptide single-chain antibody imitating a conformational epitope, apolyclonal, a monoclonal, a humanized and/or a labelled antibody.

In a preferred embodiment of the invention the neurons are afferent,efferent, intercalary, peripheral-motoric, central-motoric,preganglionic, postganglionic, and/or sensitive neurons.

The invention also relates to a method of isolating neurons, neuraland/or neuronal stem cells from a sample, wherein a recognition moleculedirected towards a protein regulated by an L1 promoter, towards anL1-associated molecule and/or towards L1 is contacted with the sample,thereby forming recognition molecule-L1 association products, and theassociation products are separated. The same definitions as in theabove-mentioned use apply to the method according to the invention, sothat reference can be made thereto. The recognition molecules directedtowards the protein regulated by an L1 promoter, towards theL1-associated molecule and/or towards L1 form association products withthe above-mentioned structures. In contrast, no association productswill be formed with structures, especially cells, included in thesample, which do not have the above-mentioned L1 equivalents. Thedifferent charge, density or size of those cells not forming anyassociation products and those representing a component of theassociation products allows separation of these different cells. Variousmethods for this purpose are well-known to those skilled in the art. Themethod of the invention can be preceded or followed by severalmechanical, physical, chemical and/or biological steps; for example, thesample can be broken up first using enzymes or simple mechanical sizereduction or specific forms of ultrasonic treatment to disintegrate themore or less intact tissue as a whole, without destroying the individualcells, particularly the neurons to be isolated or the above-mentionedstem cells.

Thus, the method results in association products which can be separated,thereby obtaining the isolated cells. In particular, it is possible toseparate the cells from the recognition molecules in a further stepwherein the association products are disintegrated. Where therecognition molecules are immobilized on a support, the isolated cellscan be removed therefrom using an enzyme. However, it can also beenvisaged not to separate the cells from the recognition molecules, e.g.chelators, particularly if these molecules have a favorable influence onthe physiology of the cells consequently, the separated associationproduct can be an isolated cell in the meaning of the invention, as isthe case with the cell which is no longer part of an association productbecause it has been separated from the recognition molecules.

Various standard publications on histology, tissue or cell culturetechnology are known to those skilled in the art, from which it can beinferred how to pretreat the sample in accordance with the cellsincluded therein, in order to disintegrate the whole sample, e.g. anembryonal mouse brain, in a way so as to allow separation of cells. Forexample, initial leaching of calcium from the tissue can be envisaged,so as to break up the cell linkages. Depending on the type of samples,initial introduction of calcium into the tissue may also be reasonablein achieving the effect of breaking up the cell linkages.

In a further step, adapting the extracellular solution in its ioncontent to the intracellular solution can be reasonable or advantageous,so that the cells would be in a better situation to survive inparticular small lesions during the isolation procedure. Depending onthe cells to be obtained, it is of course also possible not to adapt theextracellular solution in its ion content to the intracellular solution.

Furthermore, perfusion of enzymes breaking up the cell-cell linkages anddisintegrating the extracellular collagen framework possibly stabilizingthe tissue can also be envisaged. To this end, e.g. the successive useof enzymes such as trypsin and collagenase in a solution including ahigh proportion of potassium is advantageous. This step can be omittedif the sample contains no or merely an insignificant collagen framework,or if treatment with enzymes such as trypsin and/or collagenase of thecells to be isolated is undesirable, or if a high proportion ofpotassium is undesirable. However, it is also possible to envisage anenzyme treatment using a solution including a proportion of other uni-,bi- or polyvalent elements, particularly metal ions, rather than asolution including a high proportion of potassium.

In a further step, the broken up tissue can be cut and single cells canbe removed from the tissue network by mechanical stirring. At the sametime, it is possible e.g. to re-transfer the cells to be isolated intoan ionic solution of normal composition, the term normal compositionimplying that the solution ensures that pathological conditions wouldnot prevail over physiological conditions in the cells to be isolated,thereby enabling further culturing or growth or differentiation of thecells.

In a further step, pre-separation of the cells via centrifugation,sedimentation or other purification steps can be advantageous.

For example, further purification can be effected in chromatographycolumns, in a cell electrophoresis, in a FACS apparatus, or by means ofother methods of cell purification and cell isolation. However, thesesteps can also be performed in the method according to the inventionrather than in a previous procedure.

In chromatography, such as a column-chromatographic separation, it ispossible to use nylon wool, for example, on which the recognitionmolecules are immobilized, so that the cells to be isolated interactwith the wool in such a way that isolation is possible.

Furthermore, the cells can also be separated using centrifugation, forexample. More specifically, the cells can be separated using a rotatingcentrifuge, the cells being introduced via so-called bubble fillingobviously, separation can also be effected in centrifuge tubes ascommonly used. Similarly, any procedure used in cell separation, e.g. inthe preparation of concentrates, can be employed according to theinvention. This may also include cell separation by means of magneticbeads. That is, all separation methods based on differences in the celldensity, e.g. isopyknic centrifugation, the cell size, e.g. elutriationcentrifugation, the adherence properties, e.g. adsorption on plastic ornylon wool fractionation, can be employed.

In addition, separation involving magnetic particles can be used in cellseparation or isolation.

For example, such a separation can be effected using MACS or Mini-MACSor particular FACS apparatus.

Of course, all of the above-mentioned procedures can be combined withone another, i.e., it is possible to combine e.g. magnetic beadseparation with flow cytometry. However, successively employedrecognition molecules may not recognize the same epitope in each case,thereby giving rise to competition. Various ways of avoiding thisproblem or others are well-known to those skilled in the art.

Afferent, efferent, intercalary, peripheral-motoric, central-motoric,preganglionic, postganglionic neurons are isolated as neurons in apreferred embodiment of the invention.

In another preferred embodiment the neurons are adendritic, apolar,bipolar, multipolar, polyneuritic, pseudo-unipolar, unipolar and/orneurosecretory neurons.

A brain, a striatum, a neocortex, a spinal cord and/or a partialquantity thereof is employed as sample in another preferred embodimentof the invention.

Furthermore, stem cells, in particular differentiated stem cells, arepreferably used as sample, the use of neural and/or neuronal stem cellsbeing particularly preferred.

In a particularly preferred embodiment the sample comprises aneural-differentiated embryonal and/or a neural-differentiated adultstem cell.

In another preferred embodiment of the invention molecules selected fromthe group comprising an antibody, an aptamer, a lectin, an antisenseconstruct, a selective chelator, fragments or a combination thereof areemployed as recognition molecules.

In a particularly preferred fashion an antibody fragment, a single-chainantibody, a multibody, a Fab fragment, an MHC molecule, an MHC peptide,a fusion peptide, a mimicry peptide single-chain antibody imitating aconformational epitope, a polyclonal, a monoclonal, a humanized and/orlabelled antibody is used as antibody in the method according to theinvention.

In another preferred embodiment of the invention the recognitionmolecules are immobilized. Immobilization in the meaning of theinvention is understood to comprise all methods restricting the mobilityand solubility of recognition molecules or L1 equivalents by chemical,biological and/or physical means. Immobilization can be effected usingvarious methods, such as binding of recognition molecules to oneanother, or to culture dishes or separation surfaces, e.g. nylon wool,by entrapping in a network of a polymer separation matrix or byenclosure by membranes. Immobilization not only renders the recognitionmolecules reusable but also, they can easily be removed after theprocess of interaction with the biological sample. They can be employedat much higher local concentrations and in continuous flow systems.Binding or immobilization of the recognition molecules on a surface canbe effected by direct linkage to a support, in particular mediated viaspacer binding and crosslinking. According to the invention, supportbinding or crosslinking is effected particularly in an ionic/adsorptivefashion or by covalent binding. Crosslinking in the meaning of theinvention is crosslinking of recognition molecules with each other orwith other polymers. In immobilization by enclosure the recognitionmolecules are entrapped in gel structures or in membranes in such a waythat interaction and isolation of the desired cells is possible.

In another preferred embodiment of the invention the recognitionmolecules are immobilized by physical, chemical and/or biological means,by in situ synthesis, or by deposition of previously synthesizedrecognition molecules.

In an advantageous embodiment of the binding assay the recognitionmolecules are immobilized by physical, chemical and/or biological means,by in situ synthesis, or by deposition of previously synthesizedrecognition molecules on a surface. Irrespective of the type ofimmobilized recognition molecules, this can be done basically in twodifferent ways:

-   1. Deposition and immobilization of previously synthesized or    library-derived recognition molecules on well-defined positions of    an, in particular, functionalized support material. To this end,    both spotting and printing procedure can be used. Spotting is    understood to comprise procedures wherein drops of a liquid are    deposited, essentially round spots being formed as a result of    surface interaction and drying. Other printing procedures allow    deposition of the substrate in well-defined areas on the surface.-   2. In situ synthesis of recognition molecules at defined positions    of a surface, e.g. on a culture vessel or on a chromatographic    support material, by successive coupling of monomeric components for    synthesis.

In another advantageous embodiment the recognition molecules areimmobilized by contact tip printing, ring-and-pin printing andnanopipetting, bubble-jet printing, top-spot printing, microcontactprinting, micro-fluidic networks methods, photolithographic activationprocedures, photoresist lithography, electrochemical focusing and/ormicro-wet printing.

In another preferred embodiment of the invention the surface, e.g. ofthe culture or washing vessels or other equipment or materials used inseparation, is coated with poly-L-lysines, aminosilanes,aldehyde-silanes, epoxy groups, streptavidin, polylysines, silanes,reactive groups, polyacrylamide pads, immobilized nitrocellulose,activated aldehydes, agarose aldehyde groups and/or tresyl groups.Advantageously, such substrate surface treatment allows improvement ofthe binding capacity of the surface in such a way that the recognitionmolecules remain properly immobilized over a prolonged period of time.Obviously, it is also possible to treat the surface in such a way thatthe cells to be isolated have good adherence and the other componentshave less good adherence, or vice versa. Of course, surface modificationcan be effected in a broad fashion, and particularly by exposure tobiological, physical and/or chemical effects. For example, physicalexposure includes polishing, etching, scouring, sandblasting, but also,physical procedures resulting in curing, coating, finishing, coatingwith protective skins and the like. Surface treatment by biologicalexposure may comprise growth of selected cells, for example. Chemicalmodification of the surface involves e.g. treatment with acids, bases,metal oxides etc. The surface can be modified in such a way that thedetection molecules or recognition molecules have particularly goodadherence, or adherence in such a way that their activity will not beadversely modified. Naturally, surface modification of the separationmaterials or culture vessels also comprises a treatment resulting inhigher stability or improved culture conditions. Obviously, it is alsopossible to perform traditional surface modifications as in histology,such as coating with e.g. protein glycerol, polylysine, activateddextranes and/or bichromated gelatin.

In another preferred embodiment of the invention immune complexes areformed as association products. More specifically, immune complexes areformed at an optimum concentration ratio between L1, L1-associatedmolecule or protein regulated by an L1 promoter and the recognitionmolecules. In the meaning of the invention it is possible to distinguishbetween soluble and circulating immune complexes.

In a preferred embodiment of the invention the immune complexes areantibody-antigen complexes or lectin-antigen complexes.

In the meaning of the invention the antigen is the L1 cell adhesionprotein, the L1-associated molecule and/or the protein regulated by anL1 promoter, or the L1 equivalents.

In particular, lectins can be structures undergoing highly specificinteraction with molecules associated with L1 in such a way thatisolation of the desired cells is possible. For example, such lectinscan be isolated from the following organisms: Abrus precatorius, Adeniadigitata, Agaricus bisporus, Aleuria aurantia, Amaranthus caudatus,Amphicarpaea biacteata, Anguilla anguilla, Arachis hypogaea, Artocarpusintegrifolia, Bauhinia purpurea alba, Brachypodium sylvaticum, Canavaliaensiformis, Carcino scorpius rotunda canda, Cicer arietinum, Codiumfragile, Crotalaria juncea, Cytisus sessilifolius, Datura stramonium,Dioclea grandiflora, Dolichos biflorus, Erythrina coralldendron,Erythrina cristagalli, Euonymos europaea, Galanthus nivalis, Glycinemax, Griffonia (Bandeiraea) simplicifolia, Helix aspersa, Helix pomatia,Hippeastrum hybrid, Hordeum vulgare, Hura crepitaus, Latyrus odoratus,Latyrus sativus, Latyrus tingitanus, Lens culinaris, Limax glavus,Limulus polyphemus, Lotus tetragonolobus, Lycopersicon esculentum,Maackia amurensis, Maclura pomifera, Macrotyloma axillare, Momordicacharantia, Narcissus pseudonarcissus, Oryza sativa, Phaseolus coccineus,Phaseolus limensis, Phaseolus vulgaris, Phytolacca americana, Pisumsativum, Phosphocarpus tetragonolobus, Pseudomonas aeruginosa, Ricinuscommunis, Sambucus nigra, Secale cereale, Solanum tuberosum, Sophorajaponica, Triticum vulgaris, Tritrichomonas mobilensis (Protozoa), Ulexeuropaeus, Vicia cracca, vicia ervilia, Vicia graminea, Vicia faba,Vicia sativa, Vicia villosa, Viscum album, Wisteria floribunda, andalso, the following human lectins, particularly produced on arecombinant route, can be used: E-selectin (human, recombinant)L-selectin (human, recombinant) and/or P-selectin (human, recombinant),as well as galectins (human).

The antibodies can be obtained from any organism which, as a result ofdealing with antigenic immunologic recognition substances, is capable offorming in particular antibodies directed towards the latter.

In another preferred embodiment of the invention the associationproducts are antisense hybrids, with DNA-DNA, RNA-RNA and/or RNA-DNAantisense hybrids being preferred. Hybridization in the meaning of theinvention is sequence-dependent pairing of single-stranded RNA or DNAmolecules to form an RNA/DNA hybrid. The term antisense symbolizes thecomplementary structure of each single strand with respect to anothersingle strand. A variety of antisense techniques are known to thoseskilled in the art, particularly in the field of electrochemicaldetection of oligonucleotides. A person skilled in the art will alsoknow how to employ these antisense constructs, e.g. in immobilized form,in order to use them in the isolation of the desired cells. In thisevent, the antisense constructs would serve as so-called scavengersequences which are bound to a surface, for example, e.g. a culturedish, and are complementary to part of a nucleic acid structure encodingan. L1-associated molecule, a protein regulated by an L1 promoter, or anL1. For example, it can also be envisaged to make initial use of theantisense constructs to detect the corresponding structures and use themSubsequently on a protein level, e.g. together with antibodies, toisolate the desired cells.

In another preferred embodiment of the invention separation of thedesired cells is effected via different density, size and/or charge ofthe neurons, of the neuronal and/or neural stem cells, being present inthe form of association products, and of components of the samples notcontaining any association products. The term “not containing anyassociation products” refers to components of a sample which essentiallydo not comprise any L1 cell adhesion proteins, L1-associated moleculesand/or proteins regulated by an L1 promoter, or exhibit concentrationsof these structures at such low levels that isolation via use thereof isnot possible. However, those components of a sample which bear thesestructures will form L1-recognition molecule association products withthe recognition sequences, the sample including association products aswell as proteins, cells, tissue fragments and other biological unitslacking such association products. The above two groups differ in theirdensity, size and/or charge.

In a preferred embodiment of the invention said association products areseparated or isolated using affinity chromatography, cytolysis, FACS,density gradient separation, adhesion, agglutination, rosette formationand/or cell electrophoresis. With reference to a non-limiting example,this principle will be illustrated as follows: for example, it can beenvisaged that the recognition molecules are antibodies immobilized on aPetri dish. A cell suspension including neurons, among other things, canbe placed on this Petri dish. For example, these neurons can be isolatedon the Petri dish as so-called L1-positive cells via formation ofassociation products. However, it can also be envisaged to use therecognition molecules in the form of a column material, e.g. in achromatographic column, or to bind the recognition molecules to such acolumn material in order to separate a cell suspension by means of saidcolumn material, in which case the L1-positive cells will adhere to thecolumn material and all non-L1-positive cells will pass through thecolumn. The bound cells are then removed from the column material usingspecific elution procedures.

In the meaning of the invention L1-positive cells are all cells havingL1 or L1-associated molecules or proteins regulated by an L1 promoter ata concentration or with a structure allowing isolation of neurons,neural and/or neuronal stem cells from a sample via use thereof. Thoseskilled in the art are familiar with other methods of cell separation,e.g. cell separation using magnetic beads or the “panning” procedure orFACS, adsorption on plastic, nylon wool fractionation, various forms ofcentrifugation utilizing the cell size or cell density as distinguishingfeature of the cells present in the form of association products ornon-associated. In magnetic bead cell separation the L1 equivalentsserve as surface markers, the specific recognition molecules being usedin the form of magnetic bead-coupled recognition molecules.

In another preferred embodiment the sample is initially pretreated withan SH-proteinase. In a preferred fashion said SH-proteinase is papain.

In another preferred embodiment initial depletion of microglia cells inthe sample is envisaged. For example, such depletion can beeffected—using one, two or more subtraction plates—by adding thesuspension with e.g. anti-macrophage antibodies and successively placingthe suspension on two dishes coated with secondary antibodies. Themicroglia cells, for example, will adhere to the dishes because theybind to Fc fragments of the antibodies.

In another preferred embodiment removal with trypsin of the isolatedneurons, neural and/or neuronal stem cells from the immobilizedrecognition molecules or from the surfaces of the culture or separationmaterials is envisaged. For example, L1-positive cells are removed fromanti-L1 antibody-coated culture dishes by treatment with trypsin.

According to another embodiment of the invention, the cells thusisolated are then plated on cell culture dishes coated withpoly-D-lysine and/or laminin.

The invention also relates to purified neuron, neural and/or neuronalstem cell populations, particularly those obtained by means of themethod according to the invention; accordingly, the invention alsorelates to cell populations having appropriate degrees of purity, whichcan be obtained in another way. Preferred are those cell populationswith a purity of more than 90%. That is, the cells are present withoutthe material associated with the cells in their natural state or withpart thereof at maximum. Based on the weight or volume of the totalcells in a particular sample, the purified cells account for at least90%. In a particularly preferred fashion the isolated cells areessentially free of other cells, but also of proteins, lipids,carbohydrates, or other substances associated with the cells in such away that a person skilled in the art would not regard them as cells ofhigh purity or as isolated cells. Of course, this does not mean thatcells could not be associated with proteins, lipids, carbohydrates,particularly where molecules are concerned that are synthesized by theneurons, neural and/or neuronal stem cells themselves. “Essentiallyisolated” or “essentially free of” means that the cells are at least90%, preferably at least 95%, preferably at least 97%, more preferablyat least 98%, and especially preferably more than 99% free of othercells naturally associated therewith.

The invention also relates to the above-mentioned cell populations withhigh purity.

In a preferred embodiment of the invention the cells are available witha purity of 95%. In another, particularly preferred embodiment of theinvention the cells are available with a purity of more than 97%. Inanother particularly preferred embodiment of the invention the cells areavailable with a purity of more than 98%, and in yet anotherparticularly preferred embodiment of the invention the cells areavailable with a purity of more than 99%.

The invention also relates to a purification kit comprising arecognition molecule directed towards a protein regulated by an L1promoter, towards an L1-associated molecule and/or towards an L1 celladhesion protein. Using said kit, a person skilled in the art is able toisolate the above-mentioned cells which are to be purified. Isolation ispreferably performed using the method described above. Owing to theteaching that has been provided, a person skilled in the art can isolatethe cells using routine tests or equivalent uses, said routineprocedures and equivalents being hereby incorporated in the teaching ofthe present patent application.

The invention also relates to a microarray comprising a recognitionmolecule directed towards a protein regulated by an L1 promoter, towardsan L1-associated molecule and/or towards an L1 cell adhesion protein.

Without intending to be limiting, the invention will be explained inmore detail with reference to the following examples.

An immunopanning procedure was established, allowing purification ofneurons from hippocampi of postnatal mice via the neuron-specific L1antigen (Rathjen & Schachner, 1984). FIG. 1 illustrates the procedurefor the preparation of hippocampus neurons. Initially, a suspension ofdissociated hippocampi was depleted in microglia cells, using twosubtraction plates. Thereafter, L1-positive cells were isolated on aselection plate. Immunopanning via L1 allowed isolation of an average of294,000±37,000 cells (n=11 work-ups) from the hippocampi of 10-15postnatal mice (day 6-7).

Using said immunopanning, it was possible to isolate a large number ofcells. Now, it was important to see whether these cells were neurons andwhether the cultures would also contain non-neuronal cells. After 2 daysin culture, all cells showed neuron-like morphology and immunoreactivityto antibodies against the neuron markers tau, MAP2, neurofilament, andsynapsin (FIG. 2). Moreover, the cells had axonal growth cones.Differentiation of neurites into axons can be derived from the fact thatsome appendices could be stained for the tau axon marker, but not forthe marker of dendrites, MAP2 (FIG. 2 a). Furthermore, some growth coneshad synapsin-positive granules typical of growth cones of axons (FIG. 2b). These results demonstrate that the isolated cells were neurons. Itwas possible to show that 17±2% (mean value±standard error; n=4work-ups) of all neurons contained in the suspension of dissociatedhippocampi could be isolated on the selection plate.

Next, the cultures were investigated for the presence of cell typesother than neurons. To this end, immunocytochemical staining for markersof a number of non-neuronal cell types was performed (FIG. 3). Themacroglia cells were detected by staining for the specific markers2′,3′-cyclonucleotide-3′-phosphohydrolase (CNPase) and O4 foroligodendrocytes and precursor cells thereof, and for glial fibrillaryacidic protein (GFAP) for astrocytes. Since not all of the astrocytes ofthe hippocampus express GFAP, staining was also performed for S100βwhich is another astrocyte marker. Fibroblasts were detected viafibronectin, microglia cells via Griffonia simplicifolia lectin Iisolectin B4 (GSL I-B4). Control experiments showed that thesenon-neuronal cell types could be detected reliably by means of theemployed markers in one to two-day-old control cultures containing amixed cell population of dissociated hippocampi from mice (FIG. 3 b).Cultures of neurons isolated via L1 immunopanning did not include anyastrocytes. Oligodendrocytes were detected in very low amounts of <0.1%(n=4 cultures; FIG. 3 a). Also, the cultures were free of fibroblasts ormicroglia cells (n 3 cultures, 1363 cells).

These results show that cell populations comprising >99.9% ofhippocampus neurons can be obtained by means of the new immunopanningprocedure.

Also, cell isolation via L1 was found suitable in the purification ofneural-differentiated embryonal stem cells for neurons. Fibers growingout of embryoid bodies (EBs) were shown to express L1 afterdifferentiation by retinolic acid. Using immunopanning of dissociatedEBs, it was possible to isolate a large number of cells which showedneuron-like morphology and immunoreactivity to the MAP2 neuron markerafter culturing for one week. Only aminor proportion of the cells (<99%)did not have appendices and/or failed to express MAP2.

In summary, it can be stated that the method according to the inventionallows highly effective isolation of neurons from mixed cellpopulations. Using this method, it is possible to accumulate neuronswith high purity either from postnatal brain tissue or fromneural-differentiated stem cells. Neurons purified in this fashion aresuitable for use in investigating the conditions of neuronal growth anddifferentiation. Compared to mixed cell populations, they also offeradvantages in transplantations for the treatment of neurodegenerativediseases.

FIG. 1 Purification of Hippocampus Neurons

Hippocampi from 10-15 postnatal mice (day 6-7) were pretreated withpapain, followed by mechanical dissociation. Initially, the cellsuspension was depleted in microglia cells by adding the suspension withe.g. anti-macrophage antibodies and successively placing the suspensionon two dishes coated with secondary antibodies. In this step themicroglia cells—binding to the Fc fragments of the antibodies—remainedadhered to the dishes. Subsequently, L1-positive cells were isolatedfrom the cell suspension, using a dish coated with secondary antibodiesand anti-L1 antibodies. The cells were removed from the dish bytreatment with trypsin and subsequently plated on cell culture dishescoated with poly-D-lysine and laminin.

FIG. 2 Neuron-Like Morphology and Immunoreactivity to Antibodies AgainstNeuronal Markers in Isolated Hippocampus Cells

a Phase contrast image of a two-day-old culture. The cells already haveneurite-like appendices with growth cones.

b, c Immunocytochemical double-labelling for neuron markers MAP2 and tau(a) and neurofilament and synapsin (b). In stained cultures all cellsrecognizable in the phase contrast were positive for the respectiveneuron marker. The arrows denote axonal growth cones. Bar=10 μm (a), 40μm (b, c)

FIG. 3 Purity of Cultures of Isolated Hippocampus Neurons

a The proportion of macroglia cells and neurons relative to the overallnumber of cells identified via fluorescence-labelled nuclei isillustrated. To detect macroglia cells, two-day-old cultures of purifiedhippocampus neurons were simultaneously stained immunocytochemicallywith antibodies against GFAP, S100β, O4 and CNPase. Two out of 2451cells (n=4 cultures) were identified as oligodendrocytes. The error barsrepresent standard deviations.

b Control stainings showed that the markers used reliably detect therespective cell types in two-day-old mixed cultures of dissociated mousehippocampi. Bar 20 μm.

1-36. (canceled)
 37. Use of an L1 cell adhesion protein for theisolation of neurons, neural and/or neuronal stem cells from a sample.38. The use according to claim 37, characterized in that the neurons areafferent, efferent, intercalary, peripheral-motoric, central-motoric,preganglionic, post-ganglionic, and/or sensitive neurons.
 39. A methodfor the isolation of neurons, neural and/or neuronal stem cells from asample, characterized in that an antibody directed against L1 iscontacted with the sample, thereby forming antibody-L1 associationproducts, and the association products are separated.
 40. The methodaccording to claim 39, characterized in that afferent, efferent,intercalary, peripheral-motoric, central-motoric, preganglionic,postganglionic, and/or sensitive neurons are isolated as neurons. 41.The method according to claim 40, characterized in that the neurons areadendritic, apolar, bipolar, multipolar, polyneuritic, pseudo-unipolar,unipolar and/or neurosecretory neurons.
 42. The method according toclaim 39, characterized in tht a brain, a striatum, a neocortex, aspinal cord and/or a partial quantity thereof is employed as sample. 43.The method according to claim 39, characterized in that stem cells areused as sample.
 44. The method according to claim 43, characterized inthat neural and/or neuronal stem cells are used as sample.
 45. Themethod according to claim 44, characterized in thatneural-differentiated embryonal and/or neural-differentiated adult stemcells are used as sample.
 46. The method according to claim 39,characterized in that an antibody fragment, a single-chain antibody, amulti-body, a Fab fragment, an MHC molecule, an MHC peptide, a fusionpeptide, a mimicry peptide single-chain antibody imitating aconformational epitope, a polyclonal, a monoclonal, a humanized and/orlabeled antibody is used as antibody.
 47. The method according to claim37, characterized in that the antibodies are immobilized.
 48. The methodaccording to claim 47, characterized in that the antibodies areimmobilized by physical, chemical and/or biological means, by in situsynthesis, or by deposition of previously synthesized antibodies. 49.The method according to claim 48, characterized in that the antibodiesare immobilized by contact tip printing, ring-and-in printing,nanopipetting, bubble-jet printing, top-spot printing, microcontactprinting, microfluidic networks methods, photolithographic activationprocedures, photoresist lithography, electrochemical focusing and/ormicro-wet printing.
 50. The method according to claim 48, characterizedin that an immobilization surface is coated with poly-L-lysines,aminosilanes, aldehyde-silanes, epoxy groups, streptavidin, reactivegroups, polyacrylamide pads, immobilized nitrocellulose, activatedaldehydes, agarose aldehyde groups and/or tresyl groups.
 51. The methodaccording to claim 39, characterized in that immune complexes are formedas association products.
 52. The method according to claim 37,characterized in that separation is effected via different density, sizeand/or charge of the association products of components of the samplenot comprising any association products.
 53. The method according toclaim 52, characterized in that separation is effected using affinitychromatography, cytolysis, FACs, density gradient separation, adhesion,agglutination, rosette formation and/or cell electrophoresis.
 54. Themethod according to claim 37, characterized in that the sample ispretreated with an SH-proteinase.
 55. The method according to claim 54,characterized in that the SH-proteinase is papain.
 56. The methodaccording to claim 37, characterized in that the sample is initiallydepleted in microglia cells.
 57. The method according to claim 37,characterized in that the isolated neurons, neural and/or neuronal stemcells are removed from the immobilized antibodies by means of trypsin.58. The method according to claim 37, characterized in that the isolatedneurons, neural and/or neuronal stem cells are plated on cell culturedishes coated with poly-D-lysine and/or laminin.
 59. Neurons, neuraland/or neuronal stem cell population which can be obtained by means of amethod according to claim
 39. 60. The neurons, neural and/or neuronalstem cell population according to claim 59, characterized in that thecells are available with a purity of more than 90%.
 61. The neurons,neural and/or neuronal stem cell population according to claim 60,characterized in that the cells are available with a purity of more than95%.
 62. The neurons, neural and/or neuronal stem cell populationaccording to claim 61, characterized in that the cells are availablewith a purity of more than 97%.
 63. The neurons, neural and/or neuronalstem cell population according to claim 62, characterized in that thecells are available with a purity of more than 98%.
 64. The neurons,neural and/or neuronal stem cell population according to claim 63,characterized in that the cells are available with a purity of more than99%.
 65. A purification kit comprising an antibody directed against L1.66. A microarray comprising an antibody directed against L1.